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Sequencing and analysis of Phymatotrichopsis omnivora the fungal causative agent of root rot in plants Simone Macmil Dr. Roe’s Laboratory. Background. Phymatotrichopsis omnivora = Phymatotrichum omnivorum Recalcitrant soilborne fungus Deep, long-lived inoculum

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Sequencing and analysis of Phymatotrichopsis omnivora

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Sequencing and analysis of phymatotrichopsis omnivora

Sequencing and analysis of Phymatotrichopsis omnivora

the fungal causative agent of root rot in plants

Simone Macmil

Dr. Roe’s Laboratory


Sequencing and analysis of phymatotrichopsis omnivora

Background

  • Phymatotrichopsis omnivora

    =Phymatotrichum omnivorum

  • Recalcitrant soilborne fungus

    • Deep, long-lived inoculum

    • Limited to alkaline, calcareous soils of SW USA and N Mexico

  • Root rot of >2000 dicots

    • Monocots immune

  • Belongs to the family of Ascomycetes.


Sequencing and analysis of phymatotrichopsis omnivora

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Phylogeography of P. omnivora ITS Haplotypes


Sequencing and analysis of phymatotrichopsis omnivora

Life cycle of P.omnivora

Fungal hyphae

Plant infection

Blocked

Vegetative Stage: Fungal growth in soil occurs as fungal hyphae.

Moisture

Sclerotia

Spore Mat

Plant death

Moisture

Sclerotial Stage: resting structures developed deep underground.


How big is this genome

How big is this genome?

“An active parasexual cycle combined with the presence of many possible nuclei within individual hyphal cells and within a mycelium might help to explain the survival and consequent genetic flexibility of P.omnivorum, despite its lack of a known sexual cycle or functional asexual spore stage”.

Hosford R & Gries. (1966) The nuclei and parasexuality in Phymatotrichumomnivorum. Amer.J. Bot53,570-579


Sequencing and analysis of phymatotrichopsis omnivora

Phymatotrichopsis omnivora CHEF analysis (Young Lab)

5.7 Mb

4.6 Mb

3.5 Mb

P.omnivora shows diffuse banding pattern characeristic of aneuploid heterokaryotic chromosomes


Sequencing and analysis of phymatotrichopsis omnivora

P. omnivora telomeres

Po

Po

Po

Po

OKAlf8

OKAlf8

E2368

E2368

NFOK

NFOK

OKAlf8

OKAlf8

E2368

E2368

NFOK

NFOK

Alf8

Alf8

Alf8

Alf8

23.0

23.0

6.5

2.3

EcoRI

BglII

Probed with Tubulin2

0.5

BglII

EcoRI

Probed with (TTAGGG)6


Sequencing and analysis of phymatotrichopsis omnivora

Multinucleate, heterokaryotic hyphae of P. omnivora

  • 3-20 nuclei per young hyphal cells

  • Frequent hyphal fusions


Overview of 454 dna preparation protocol

Biotin

Overview of 454 DNA preparation protocol

Nebulization

Quantitate on Caliper AMS-90

DNA End Repair

5’

3’

3’

5’

5’

3’

Adaptor Ligation (A&B)

3’

5’

5’

3’

DNA End Repair

3’

5’


Overview of 454 dna preparation protocol1

Overview of 454 DNA preparation protocol

  • Emulsify beads and PCR reagents in water-in-oil microreactors

    • - “B” primer is in solution and attached to capture bead

    • - “A” primer is biotinylated

  • emPCR Amplification

  • Break Microreactors

  • Enrich for DNA positive beads

  • Load onto 454 Plates

Before PCR

After PCR


Pyrosequencing

T

C

G

A

T

dNTP Base Addition

PP

i

Pyrosequencing

Bead

dTTP

Polymerase

A A T C G G C A T G C T A A A A G T C A

T

APS

Annealed Primer

Sulfurylase

Luciferase

ATP

luciferin

Light + oxy luciferin


P omnivora assembly statistics newbler assembly ver 2 0

P.omnivora assembly statistics (Newbler assembly ver 2.0)


P omnivora genome analysis schema

454

P.omnivora genome analysis schema

ABI 3730

Assembly with Newbler

Predict genes using FgenesH,

Predict tRNAs using tRNAScanSE

Homology search against GenBank

Metabolic Reconstruction using KEGG, KOG and COGEME


Repeats found in p omnivora searched against repbase

Repeats found in P.omnivora searchedagainst Repbase

Interspersed Repeats


Distribution of metabolic processes from proteins predicted in genomic contigs

Distribution of Metabolic processes (from proteins predicted in genomic contigs)


Sequencing and analysis of phymatotrichopsis omnivora

Relative numbers of P.omnivora, S.cerevisae, and N.crassa transporters in various families


P omnivora transport proteins

P.omnivora Transport Proteins

  • 28 drug efflux proteins

  • 19 calcium transporters

  • Transport proteins significant to its lifestyle

    • Bcmfs1, DHA14-like major facilitator superfamily multidrug transporter involved in protection against natural toxins and fungicides

    • Arsenite-translocating ATPases


Paired end sequencing on the 454 of chef gel extracted and amplified chromosomes

^^^^^^

^^^^^^

Paired-end sequencing on the 454 of CHEF gel extracted and amplified chromosomes

EcoRI

EcoRI

EcoRI

^^^^^^

Excise gel bands containing chromosomal DNA

Methylation

Ligation of sticky ends and Circularization

Chromosomal DNA extraction and isothermal amplification using the Multiple Displacement Amplification Reaction

Hairpin adaptor ligation of ends

Nebulization

^^^^^^

Generation of 454 shotgun library

and sequencing

Ecor I digestion

Hydroshear amplified DNA to 2-4 Kb pieces


Distribution of cellular processes in chromosomes 2 and 3

Distribution of cellular processes in chromosomes 2 and 3


Distribution of cellular processes in chromosomes 4 and 5

Distribution of cellular processes in chromosomes 4 and 5


Est expressed sequence tag sequencing

EST (Expressed Sequence Tag) Sequencing

Construct the 454 Library and Sequence

Isolate total RNA


Sequencing and analysis of phymatotrichopsis omnivora

Overview of EST analysis - Draft

Contigs + singlets

BlastX against KEGG and COG databases

extract_kegg_cogs_1_pl

Metabolic reconstruction

ESTanalysis_script2_FZN2_pl

Overall Expression Profile

Using COG’s schema and KEGG’s metabolism

Contigs and Singlets with No Hit

tBlastX against EST

Append the Overall Expression File


Ests studies from different life stages of p omnivora

ESTs studies from different life stages of P.omnivora


Gene expression of known function genes in vegetative mycelia

Gene Expression of Known Function genes in vegetative mycelia

84,708 reads were sequenced using the GS FLX.

High number of house keeping genes were found to be expressed


Sequencing and analysis of phymatotrichopsis omnivora

Gene Expression of Known Function genes in vegetative mycelia in response to stress (Carbon and Nitrogen starvation)

10,720 reads were sequenced using the 454 GS 20

High number of mitochondrial genes expressed along with genes involved in gluconeogenesis and proteolysis.


Gene expression of known function genes in sclerotia resting structures

Gene Expression of Known Function genes in Sclerotia (resting structures)

60,493 reads were sequenced using the 454 GS 20

High number of genes involved in fatty acid and carbohydrate metabolism


Gene expression in spore mat that consists of conidia asexual spores

Gene Expression in Spore Mat(that consists of conidia /asexual spores)

61,702 reads were sequenced using the 454 GS 20

High number of genes involved in chromatin remodelling and conidial development were expressed


Sequencing and analysis of phymatotrichopsis omnivora

Comparison of Gene Expression of P.omnivora mycelia during Interaction with the Host vs non-Host Root Exudate


Sequencing and analysis of phymatotrichopsis omnivora

Comparison of gene expression of P.omnivora mycelia during interaction with host vs non-host root exudate


Cdna analysis and conclusions

cDNA Analysis and Conclusions

  • Energy metabolism was observed for each stage in the life cycle.

  • In response to stress, fungal mycelia resort to gluconeogenesis and protein degradation.

  • Sclerotial resting structures were found to be mainly involved in carbohydrate metabolism and fatty acid biosynthesis.

  • Spore mats were found to be metabolically active, histones H3& H4 involved in the condensation of chromosomes were observed.

  • Genes involved in lipid biosynthesis, polyketide biosynthesis and signal transduction were found to be expressed only during host-root exudate interaction hence account for putative pathogenicity factors of P.omnivora.

  • Genes involved in butanoate metabolism, porphyrin & ubiquinone biosynthesis were found during interaction with non-host root exudate only and may contribute to the saprophytic life style of the fungus.


Sequencing and analysis of phymatotrichopsis omnivora

Disease and Virulence genes found in Genomic contigs and ESTs in response to Host and non-host Root Exudate Interaction

  • Similar to delta latrotoxin gene from Bortryotina

  • Pathogensis related Snod Protein1 (detected in EST response to non-host root exudate)

  • Superoxide-generating NADPH oxidase

  • CAP20-like protein (involved in virulence, penetration, and appressorium formation in Blumeria graminis)

  • Homologue of M. grisea pathogenicity protein

  • Structural toxin protein homologue (detected in EST response to host root exudate)

  • Superoxide generating NADPH oxidase cytosolic protein


Summary conclusions

Summary & Conclusions

  • The fungus P.omnivora has numerous nuclei in it’s mycelia, high number of repeat elements with a genome size of ~74 Mb characteristic of an obligate heterokaryote.

  • Preliminary assembly and analysis of the genome revealed high number of transporter genes.

    • The high number of ABC type transporters most likely confers resistance to fungicides, heavy metals and other plant toxins.

    • The high number of calcium transporters may account for P. omnivora’s ability to live in calcareous soils.

  • The expression of specific cDNAs (ESTs) under different conditions reveals specific mRNAs that are involved with each unique stage of fungal growth and development cycle.


Acknowledgements

Acknowledgements

  • Dr. Bruce A. Roe

  • Dr. F. Najar, Steve Kenton

  • Graham Wiley, ChumMei Qu, Ping Wang, Yanbo Xing, Doug White

  • All other members of Dr. Roe’s lab

  • Dr. C. Young and Dr. S.Marek at the Noble Foundation and OSU repectively

  • Graduate Committee members: Dr. P.Cook, Dr.A. West, Dr. P. Klebba, Dr. K. Duncan, Dr. C. Rice,

  • Oklahoma Dept. of Agriculture for funding the Oklahoma Consortium for Legume Research


Sequencing and analysis of phymatotrichopsis omnivora

Devol, OK


Sequencing and analysis of phymatotrichopsis omnivora

Library ssDNA

3’

5’

B

A

Emulsify with

A&B primers,

A’ is Biotinylated

on 5’

16B’=A’

5’

3’

5’

3’

3’

5’

B’

A’

3’

5’

94oC Hot Start

5’

3’

5’

3’

3’

5’

B’

A’


Sequencing and analysis of phymatotrichopsis omnivora

3’

5’

Free B’ Extends

Along Free Template

5’

Free Biotinylated

A’ Extends

5’

[

]

X10^6

emPCR Final Result

5’

3’


Sequencing and analysis of phymatotrichopsis omnivora

[

]

emPCR Final Result

X10^6

5’

3’

Bind to

Streptavidin Coated

Enrichment Bead

Use Magnet to

Sequester Beads

Then Melt the DNA

Anneal Sequencing

Primer

3’

5’

Load on 454


Sequencing and analysis of phymatotrichopsis omnivora

700 bp

IGS

ITS2

ITS1

1700 bp

1400 bp

5.8S

SSU (18S) RNA

LSU (28S) RNA

2900 bp

  • Molecular Systematics of P. omnivoraMarek, Hansen, Romanish, and Thorn (2008)

  • 129 isolates of Phymatotrichopsis omnivora

  • Genes sequenced

  • Nuclear Ribosomal Genes (rDNA)

  • RNA pol II subunit-2 (rpb2)

2.9Kb


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